The distribution of fitness effects (DFE) of new mutations is of fundamental importance in evolutionary biology and has practical applications in biomedicine, including complex disease and cancer. Several methods exist by which the DFE can be estimated, each of which has limitations. Indirect estimates derived from standing genetic variation provide information about the DFE of mutations under very weak to moderate selection but provide little information about strongly selected mutations. Conversely, estimates of the DFE from experimental data provide information about relatively strongly selected mutations but can only provide information about weakly selected mutations in aggregate. A comprehensive characterization of the full DFE must integrate both kinds of information. Importantly, in humans the fitness effects of all but the mos highly deleterious mutations cannot be estimated directly. Direct estimates of the DFE from model organisms can inform efforts to model human population genetic processes, as well as advance our understanding of evolution in general. The nematode C. elegans provides the ideal model system to in which to experimentally characterize the DFE: its short generation time, unparalleled genetic resources and ease with which stocks can be cryopreserved make it unique among multicellular organisms. In Aims 1 and 2, stocks of C. elegans that have accumulated mutations for 250 generations under minimal natural selection will be employed to estimate the DFE, combining the methods of classical quantitative genetics with high-throughput genotyping and phenotyping. The goals of Aim 1 are (1) characterize the genome-wide mutation rate by sequencing a set of mutation accumulation (MA) lines, and (2) obtain an accurate estimate of the decline in fitness with MA by large-particle flow cytometry (aka a worm sorter) to measure lifetime reproduction of a very large number of individuals. MA lines will be mated to the unmutated ancestor to generate F1 heterozygotes, which will be similarly characterized for fitness. In Aim 2, a large panel of Recombinant Inbred Advanced Intercross Lines (RIAILs) will be constructed from a cross of two fully-sequenced MA lines. Each RIAIL will be genotyped at each mutant locus and individual mutations can be treated as quantitative trait loci (QTL), from which the homozygous DFE can be inferred statistically. To estimate the heterozygous DFE, each RIAIL will be mated to the ancestor of the MA lines and fitness determined in the F1 offspring. Further, it will be apparent if overdominant mutations (i.e., heterozygote advantage) are present; unambiguous evidence for true overdominance is almost non-existent. In both Aims 1 and 2, fitness will be assessed under several relevant environmental conditions. In Aim 3, the DFE will be determined statistically from the site-frequency spectrum in a set of 162 wild isolates of C. elegans. The proposed work will provide the most comprehensive characterization of the DFE in any multicellular organism, and will inform any study in applied population genetics.